Search Results (36)

The bacterial flagellum, a complex nanomachine composed of numerous proteins, is utilized by bacteria for swimming in various environments and plays a crucial role in their survival and infection. The flagellar motor is composed of a rotor and stator complexes, with each stator unit functioning as an ion channel that converts flow from outside of cell membrane into rotational motion. Paenibacillus sp. TCA20 was discovered in a hot spring, and a structural analysis was conducted on the stator complex using cryo-electron microscopy to elucidate its function. Two of the three structures (Classes 1 and 3) were found to have structural properties typical for other stator complexes. In contrast, in Class 2 structures, the pentamer ring of the A subunits forms a C-shape, with lauryl maltose neopentyl glycol (LMNG) bound to the periplasmic side of the interface between the A and B subunits. This interface is conserved in all stator complexes, suggesting that hydrophobic ligands and lipids can bind to this interface, a feature that could potentially be utilized in the development of novel antibiotics aimed at regulating cell motility and infection. Full article

In this study, we investigated the anti-fatigue effects of black ginseng ginsenosides using exercise performance tests, serum analyses, and gene expression profiling. No significant differences in dietary intake or body weight were observed between groups. The low-dose black ginseng (LBG) group showed no significant improvements in swimming and rotating rod tests. In contrast, the medium (MBG)- and high-dose (HBG) groups showed notable increases in swimming time and significant improvements in the rotating rod test. All treatment groups exhibited longer running times, particularly the HBG group. Serum analysis revealed increased muscle and hepatic glycogen, catalase, and lactate dehydrogenase levels in the MBG and HBG groups, whereas lactate, lipid peroxide, and superoxide dismutase levels were decreased. Additionally, gene expression analysis showed significant upregulation of key antioxidant and mitochondrial function genes, including those encoding cationic amino acid transporter 2, stearoyl-CoA desaturase-2, nuclear respiratory factor 1, nuclear factor erythroid 2-related factor 2, mitochondrial transcription factor A, cytochrome c oxidase II, and NADH–ubiquinone oxidoreductase core subunit 1, particularly in the HBG group, indicating enhanced antioxidant capacity and improved mitochondrial function. These findings suggested that black ginseng ginsenosides effectively mitigated fatigue. Full article

The evolution of the translation system is a fundamental issue in the quest for the origin of life. A feasible evolutionary scenario necessitates the autonomous emergence of a protoribosome capable of catalyzing the synthesis of the initial peptides. The peptidyl transferase center (PTC) region in the modern ribosomal large subunit is believed to retain a vestige of such a prebiotic non-coded protoribosome, which would have self-assembled from random RNA chains, catalyzed peptide bond formation between arbitrary amino acids, and produced short peptides. Recently, three research groups experimentally demonstrated that several distinct dimeric constructs of protoribosome analogues, derived predicated on the approximate 2-fold rotational symmetry inherent in the PTC region, possess the ability to spontaneously fold, dimerize, and catalyze the formation of peptide bonds and of short peptides. These dimers are examined, aiming at retrieving information concerned with the characteristics of a prebiotic protoribosome. The analysis suggests preconditions for the laboratory re-creation of credible protoribosome analogues, including the preference of a heterodimer protoribosome, contradicting the common belief in the precedence of homodimers. Additionally, it derives a dynamic process which possibly played a role in the spontaneous production of the first bio-catalyzed peptides in the prebiotic world. Full article

ATP synthase inhibitory factor subunit 1 (IF1) is an inhibitory subunit of mitochondrial ATP synthase, playing a crucial role in regulating mitochondrial respiration and energetics. It is well-established that IF1 interacts with the F1 sector of ATP synthase to inhibit the reversal rotation and, thus, ATP hydrolysis. Recent evidence supports that IF1 also inhibits forward rotation or the ATP synthesis activity. Adding to the complexity, IF1 may also facilitate mitophagy and cristae formation. The implications of these complex actions of IF1 for cellular function remain obscure. In the present study, we found that IF1 expression was markedly upregulated in hypoxic MEFs relative to normoxic MEFs. We investigate how IF1 affects cellular growth and function in cultured mouse embryonic fibroblasts derived from mouse lines with systemic IF1 overexpression and knockout under normoxia and hypoxia. Cell survival and proliferation analyses revealed that IF1 overexpression exerted limited effects on cellular viability but substantially increased proliferation under normoxia, whereas it facilitated both cellular viability and proliferation under hypoxia. The absence of IF1 may have a pro-survival effect but not a proliferative one in both normoxia and hypoxia. Cellular bioenergetic analyses revealed that IF1 suppressed cellular respiration when subjected to normoxia and was even more pronounced when subjected to hypoxia with increased mitochondrial ATP production. In contrast, IF1 knockout MEFs showed markedly increased cellular respiration under both normoxia and hypoxia with little change in mitochondrial ATP. Glycolytic stress assay revealed that IF1 overexpression modestly increased glycolysis in normoxia and hypoxia. Interestingly, the absence of IF1 in MEFs led to substantial increases in glycolysis. Therefore, we conclude that IF1 mainly inhibits cellular respiration and enhances cellular glycolysis to preserve mitochondrial ATP. On the other hand, IF1 deletion can significantly facilitate cellular respiration and glycolysis without leading to mitochondrial ATP deficit. Full article

Ribosome is a major part of the protein synthesis machinery, and analysis of its structure is of paramount importance. However, the structure of ribosomes from only a limited number of organisms has been resolved to date; it especially concerns plant ribosomes and ribosomal subunits. Here, we report a high-resolution cryo-electron microscopy reconstruction of the small subunit of the Triticum aestivum (common wheat) cytoplasmic ribosome. A detailed atomic model was built that includes the majority of the rRNA and some of the protein modifications. The analysis of the obtained data revealed structural peculiarities of the 40S subunit in the monocot plant ribosome. We applied the 3D Flexible Refinement approach to analyze the internal mobility of the 40S subunit and succeeded in decomposing it into four major motions, describing rotations of the head domain and a shift in the massive rRNA expansion segment. It was shown that these motions are almost uncorrelated and that the 40S subunit is flexible enough to spontaneously adopt any conformation it takes as a part of a translating ribosome or ribosomal complex. Here, we introduce the first high-resolution structure of an isolated plant 40S subunit and the first quantitative analysis of the flexibility of small ribosomal subunits, hoping that it will help in studying various aspects of ribosome functioning. Full article

We review experimental results obtained with broadband dielectric spectroscopy concerning the relaxation times and activation energies of intramolecular conformational relaxation processes in small-molecule glass-formers. Such processes are due to the interconversion between different conformers of relatively flexible molecules, and generally involve conformational changes of flexible chain or ring moieties, or else the rigid rotation of planar groups, such as conjugated phenyl rings. Comparative analysis of molecules possessing the same (type of) functional group is carried out in order to test the possibility of assigning the dynamic conformational isomerism of given families of organic compounds to the motion of specific molecular subunits. These range from terminal halomethyl and acetyl/acetoxy groups to both rigid and flexible ring structures, such as the planar halobenzene cycles or the buckled saccharide and diazepine rings. A short section on polyesters provides a generalisation of these findings to synthetic macromolecules. Full article

The CCT/TRiC complex is a type II chaperonin that undergoes ATP-driven conformational changes during its functional cycle. Structural studies have provided valuable insights into the mechanism of this process, but real-time dynamics analyses of mammalian type II chaperonins are still scarce. We used diffracted X-ray tracking (DXT) to investigate the intramolecular dynamics of the CCT complex. We focused on three surface-exposed loop regions of the CCT1 subunit: the loop regions of the equatorial domain (E domain), the E and intermediate domain (I domain) juncture near the ATP-binding region, and the apical domain (A domain). Our results showed that the CCT1 subunit predominantly displayed rotational motion, with larger mean square displacement (MSD) values for twist (χ) angles compared with tilt (θ) angles. Nucleotide binding had a significant impact on the dynamics. In the absence of nucleotides, the region between the E and I domain juncture could act as a pivotal axis, allowing for greater motion of the E domain and A domain. In the presence of nucleotides, the nucleotides could wedge into the ATP-binding region, weakening the role of the region between the E and I domain juncture as the rotational axis and causing the CCT complex to adopt a more compact structure. This led to less expanded MSD curves for the E domain and A domain compared with nucleotide-absent conditions. This change may help to stabilize the functional conformation during substrate binding. This study is the first to use DXT to probe the real-time molecular dynamics of mammalian type II chaperonins at the millisecond level. Our findings provide new insights into the complex dynamics of chaperonins and their role in the functional folding cycle. Full article

Bacteriophage T4 is decorated with 155 180 Å-long fibers of the highly antigenic outer capsid protein (Hoc). In this study, we describe a near-atomic structural model of Hoc by combining cryo-electron microscopy and AlphaFold structure predictions. It consists of a conserved C-terminal capsid-binding domain attached to a string of three variable immunoglobulin (Ig)-like domains, an architecture well-preserved in hundreds of Hoc molecules found in phage genomes. Each T4-Hoc fiber attaches randomly to the center of gp23* hexameric capsomers in one of the six possible orientations, though at the vertex-proximal hexamers that deviate from 6-fold symmetry, Hoc binds in two preferred orientations related by 180° rotation. Remarkably, each Hoc fiber binds to all six subunits of the capsomer, though the interactions are greatest with three of the subunits, resulting in the off-centered attachment of the C-domain. Biochemical analyses suggest that the acidic Hoc fiber (pI, ~4–5) allows for the clustering of virions in acidic pH and dispersion in neutral/alkaline pH. Hoc appears to have evolved as a sensing device that allows the phage to navigate its movements through reversible clustering–dispersion transitions so that it reaches its destination, the host bacterium, and persists in various ecological niches such as the human/mammalian gut. Full article

F_oF₁-ATP synthases in mitochondria, in chloroplasts, and in most bacteria are proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes’ reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli F_oF₁-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled F_oF₁-ATP synthases in solution were extended up to several seconds using a confocal anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes, we revealed that ADP inhibition did not decrease the catalytic turnover of all F_oF₁-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios. Full article

Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation. Full article

V-ATPase is an important factor in synaptic vesicle acidification and is implicated in synaptic transmission. Rotation in the extra-membranous V1 sector drives proton transfer through the membrane-embedded multi-subunit V0 sector of the V-ATPase. Intra-vesicular protons are then used to drive neurotransmitter uptake by synaptic vesicles. V0a and V0c, two membrane subunits of the V0 sector, have been shown to interact with SNARE proteins, and their photo-inactivation rapidly impairs synaptic transmission. V0d, a soluble subunit of the V0 sector strongly interacts with its membrane-embedded subunits and is crucial for the canonic proton transfer activity of the V-ATPase. Our investigations show that the loop 1.2 of V0c interacts with complexin, a major partner of the SNARE machinery and that V0d1 binding to V0c inhibits this interaction, as well as V0c association with SNARE complex. The injection of recombinant V0d1 in rat superior cervical ganglion neurons rapidly reduced neurotransmission. In chromaffin cells, V0d1 overexpression and V0c silencing modified in a comparable manner several parameters of unitary exocytotic events. Our data suggest that V0c subunit promotes exocytosis via interactions with complexin and SNAREs and that this activity can be antagonized by exogenous V0d. Full article

Fusarium head blight (FHB), which is primarily caused by Fusarium graminearum, is a widespread and devastating disease of wheat. In the absence of resistant varieties, the control of FHB relies heavily on the application of fungicides, and the new generation SDHI fungicide, pydiflumetofen, has recently been registered in China for the control of FHB in wheat. The current study explored three genetically stable, highly resistant laboratory mutants (S2-4-2R, S27-3R, and S28-2R, with EC₅₀ values of 25.10, 28.57, and 19.22 μg/mL, respectively) to investigate the potential risks associated with pydiflumetofen resistance. Although the mycelial growth of the mutants differed little compared to their parental isolates, the study found that the resistant mutants exhibited significantly reduced (p < 0.05) levels of sporulation and pathogenicity, which suggests a significant fitness cost associated with pydiflumetofen resistance in F. graminearum. Sequence analysis of the Sdh target protein identified numerous amino acid substitutions in the predicted sequences of the four subunits: FgSdhA, FgSdhB, FgSdhC, and FgSdhD. Indeed, the mutants were found to have a series of substitution in multiple subunits such that all three exhibited five identical changes, including Y182F in the FgSdhA subunit; H53Q, C90S, and A94V in FgSdhB; and S31F in FgSdhC. In addition, gene expression analysis revealed that all of the FgSdh genes had significantly altered expression (p < 0.05), particularly FgSdhA and FgdhC, which exhibited remarkably low levels of expression. However, the study found no evidence of cross-resistance between pydiflumetofen and tebuconazole, fludioxonil, prochloraz, fluazinam, carbendazim, pyraclostrobin, or difenoconazole, which indicates that these fungicides, either in rotation or combination with pydiflumetofen, could mitigate the risk of resistance emerging and provide ongoing control of FHB to ensure high and stable wheat yields. Full article

Wildland fire literacy is the capacity for wildland fire professionals to understand and communicate fundamentals of fuel and fire behavior within the socio-ecological elements of the fire regime. While wildland fire literacy is best developed through education, training, and experience in wildland fire science and management, too often, development among early-career professionals is deficient in one or more aspects of full literacy. We report on a hands-on prescribed fire methods workshop designed to provide training and experience in measuring and conducting prescribed fire, with a focus on grassland ecosystems. The workshop was held in March 2022 at The Nature Conservancy’s Dunn Ranch Prairie in northern Missouri. It consisted of hands-on training and experience in measuring fuels, fire weather, and fire behavior. Prescribed fire operations training facilitated both hands-on learning and vicarious learning by rotating squad roles among several small sub-units on the first day of live fire exercises. Participants then gained experience as crew members for two larger prescribed burns (60 and 200 ha). We report here on the successes and lessons learned from the perspectives of both participants and the instructor cadre for what was widely regarded as a successful workshop. Full article

Protozoa are eukaryotic, unicellular microorganisms that have an important ecological role, are easy to handle, and grow rapidly, which makes them suitable for ecotoxicity assessment. Previous methods for locomotion tracking in protozoa are largely based on software with the drawback of high cost and/or low operation throughput. This study aimed to develop an automated pipeline to measure the locomotion activity of the ciliated protozoan Tetrahymena thermophila using a machine learning-based software, TRex, to conduct tracking. Behavioral endpoints, including the total distance, velocity, burst movement, angular velocity, meandering, and rotation movement, were derived from the coordinates of individual cells. To validate the utility, we measured the locomotor activity in either the knockout mutant of the dynein subunit DYH7 or under starvation. Significant reduction of locomotion and alteration of behavior was detected in either the dynein mutant or in the starvation condition. We also analyzed how Tetrahymena locomotion was affected by the exposure to copper sulfate and showed that our method indeed can be used to conduct a toxicity assessment in a high-throughput manner. Finally, we performed a principal component analysis and hierarchy clustering to demonstrate that our analysis could potentially differentiate altered behaviors affected by different factors. Taken together, this study offers a robust methodology for Tetrahymena locomotion tracking in a high-throughput manner for the first time. Full article

The F₁F_O-ATP synthase nanomotor synthesizes >90% of the cellular ATP of almost all living beings by rotating in the “forward” direction, but it can also consume the same ATP pools by rotating in “reverse.” To prevent futile F₁F_O-ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF₁), and chloroplasts (ε and γ disulfide) emerged to block the F₁F_O-ATPase activity selectively. In this study, we analyze how these F₁F_O-ATPase inhibitory proteins have evolved. The phylogeny of the α-proteobacterial ε showed that it diverged in its C-terminal side, thus losing both the inhibitory function and the ATP-binding/sensor motif that controls this inhibition. The losses of inhibitory function and the ATP-binding site correlate with an evolutionary divergence of non-inhibitory α-proteobacterial ε and mitochondrial δ subunits from inhibitory bacterial and chloroplastidic ε subunits. Here, we confirm the lack of inhibitory function of wild-type and C-terminal truncated ε subunits of P. denitrificans. Taken together, the data show that ζ evolved to replace ε as the primary inhibitor of the F₁F_O-ATPase of free-living α-proteobacteria. However, the ζ inhibitory function was also partially lost in some symbiotic α-proteobacteria and totally lost in some strictly parasitic α-proteobacteria such as the Rickettsiales order. Finally, we found that ζ and IF₁ likely evolved independently via convergent evolution before and after the endosymbiotic origin mitochondria, respectively. This led us to propose the ε and ζ subunits as tracer genes of the pre-endosymbiont that evolved into the actual mitochondria. Full article